Transcriptional regulation involves transcription factor (TF) binding to specific enhancer/silencer elements and recruitment of many coregulator proteins, which remodel chromatin and regulate assembly of an active transcription complex. TF binding to genomic sites and coregulator actions are selective; i.e. not every appropriate motif and available TF binding site is occupied, and the coregulators required for transcriptional regulation by a specific TF varies in a gene-specific manner. Gene-specific actions of coregulators represent unexplored physiological regulatory mechanisms. To facilitate future studies on coregulator physiological roles, we examine here the mechanisms by which coregulator Hic-5 modulates transcriptional regulation by the glucocorticoid receptor (GR) using gene-specific mechanisms. GR is activated by binding of glucocorticoid (GC) hormones and synthetic ligands; among many other physiological pathways, GR regulates inflammation and metabolism of glucose, fat, and protein. Regulation of some genes by GC and GR requires Hic-5 (Hic-5 modulated or mod genes), while regulation of other genes by GC/GR is not affected by Hic-5 depletion (Hic-5 independent or ind genes). We identified a third class of genes that are not regulated by GC/GR until Hic-5 is depleted from cells (Hic-5 blocked or block genes). Hic-5 strongly inhibits GC-induced binding of GR and chromatin remodeling on block genes; but on mod genes Hic-5 facilitates recruitment of the Mediator complex and RNA polymerase II. Thus regulation of Hic-5 expression or activity could modulate the set of genes regulated by GC, and thus the biological response, within a given cell type and between different cell types. The proposed project focuses on block genes, which provide a unique opportunity to explore novel mechanisms that control TF binding site selection. We will define specific characteristics of ind, mod, and block genes and their chromatin environment that determine gene-specific actions of Hic-5 (Aim 1). In addition, our preliminary data indicate that block genes require different chromatin remodeling complexes than ind and mod genes; we will therefore explore the hypothesis that Hic-5 selectively interferes with the chromatin remodelers required for GC regulation of the block genes (Aim 2). Finally, functional genomic analyses of GR and Hic-5 mutants will test the roles of the GR-Hic-5 interaction and of Hic-5 protein domains in differentia regulation of Hic-5 modulated and Hic-5 blocked genes (Aim 3). Our results will define novel molecular mechanisms and potentiate future exploration of Hic-5 roles in the physiology of GC and in binding site selection by TFs.